Ecdysteroid kinase in silkworm eggs : Purification and characterization

在许多昆虫物种的卵中，来自母体的卵磷脂被广泛认为是胚胎发育期间提供活性游离卵磷脂的储存形式（Makka et al., 2002）。然而，关于特异性催化游离外甾体磷酸化的酶的信息很少。本研究的目的是纯化和表征外皮甾体激酶（ATP：外皮甾体磷酸转移酶）。用[^3H]蜕皮激素的磷酸化反应检测蜕皮激素激酶。该酶位于家蚕成熟子房的细胞质组分中，通过Q Sepharose、Chelating Sepharose、Hydroxylapatite、Hitrap Q、Mono Q、5'AMP Sepharose 4B和Superdex 200pg 7步柱层析纯化，纯度约为600倍。纯化后的酶在pH 7.6时最具活性。该酶需要ATP和Mg^<2+>的存在才能表达活性。蜕皮激素激酶的表观动力学参数Km=3.30 μM（20-羟基蜕皮激素）、3.66 μM（蜕皮激素）、8.22 μM（3-羟基蜕皮激素）。目前尚不清楚在gregaria Schistocerca （Kabbouh and Rees 1991）中发现的ATP: 2-脱氧蜕皮激素22-磷酸转移酶与家蚕的蜕皮激素22-磷酸转移酶（蜕皮激素激酶）是否同源。

In eggs of many insect species, ecdysteroid-phosphates, which are of maternal origin, have widely been accepted as storage forms for supplying active free ecdysteroids during embryonic development (Makka et al., 2002). However, there is little information on the enzyme which specifically catalyzes the phosphorylation of free ecdysteroids. The purpose of this study is to purify and characterize the ecdysteroid kinase (ATP : ecdysteroid phosphotransferase).Ecdysteroid kinase was assayed by the phosphorylation reaction of [^3H] ecdysone. The enzyme, which was located in the cytosol fraction in mature ovaries of the silkworm Bombyx mori, was purified by about 600-fold to homogeneity by seven steps of column chromatography : Q Sepharose, Chelating Sepharose, Hydroxylapatite, Hitrap Q, Mono Q, 5'AMP Sepharose 4B and Superdex 200pg. The purified enzyme was most active at pH 7.6. The enzyme required the presence of both ATP and Mg^<2+> for expressing the activity. The apparent kinetic parameters for ecdysone kinase was Km=3.30 μM (20-hydroxyecdysone), 3.66 μM (ecdysone), 8.22 μM (3-epiecdysone). It is not clear at present whether ATP : 2-deoxyecdysone 22-phosphotransferase found in Schistocerca gregaria (Kabbouh and Rees 1991) and our ecdysone 22-phosphotransferase (ecdysone kinase) in Bombyx mori are homologous.